Project Summary/Abstract One of challenges in modern Neuroscience is to understand circuit mechanisms that lead to complex behaviors. Our ability to monitor neuronal activity in vivo using genetically encoded calcium indicators and various imaging/optogenetic techniques such as two-photon imaging and channelrhodopsin have helped us define real-time changes in neuronal activity and the circuit basis of behaviors. However, learning and behaviors cannot be solely explained by electrophysiological properties of single neurons and their synaptic connectivity because they are modulated by internal brain state. Therefore, we cannot fully understand diverse emotional or behavioral reactions without understanding the internal brain state. Neuromodulators have been suggested as key molecules that control brain state, but their action to neurons has not been understood at cellular resolution. We have recently developed a novel technique (named ?iTango2?) that labels and manipulates neuromodulation-sensitive neuronal populations with high spatiotemporal resolution. Using this iTango2 methodology, we would like to dissect neuromodulator circuits at individual cell levels, and their physiological implications related to complex behavior will be explored in this study. In the first aim, we will examine how sparse dopamine projection in the premotor cortex contributes to cortical circuit assembly, which may uncover cellular mechanisms of the asymmetric principle of cortical neuronal connectivity. Second, we will dissect neuromodulation signaling at subcellular resolution. This will be accomplished by creating a synapse version of iTango2, ?Syn-iTango2?. In order to identify potential cortical layer- or dendritic branch-specific mechanisms, we will perform real-time monitoring of local dendritic activation triggered by neuromodulatory inputs in brain slices as well as awake behaving animals, and concomitant structural changes such as spine formation or enlargement will be examined. In the third aim, we would like to identify the neuronal ensemble responsible for social interaction, one of the essential complex behaviors in mammals. Since iTango2 links neuromodulation signals to gene expression, we will test the sufficiency of identified circuits to social behavior. Last, we will build a large library of iTango2, so that this approach becomes broadly useful to a variety of neuroscientists. In summary, completion of this study will demonstrate fine scale of neuromodulation action in a quantitative manner rather than a simple ?ON? or ?OFF? effect of neuromodulation. Monitoring neuromodulatory effects and manipulating populations of cells with high spatial and temporal resolution would dramatically increase our knowledge of the pathways that underlie vertebrate animal behaviors.